Gelatin microcarrier high-efficiency lysis solution based on three-enzyme cooperation and ion activation mechanism and application thereof

Abstract

The application belongs to the technical field of cell culture and relates to a gelatin microcarrier high-efficiency lysis solution based on a three-enzyme synergistic and ion activation mechanism and application thereof.A gelatin microcarrier high-efficiency lysis solution comprises a complex enzyme and a buffer system.The lysis solution has strong pertinence, high lysis efficiency, is suitable for microcarriers made of collagen or gelatin, avoids direct exposure of cells to strong enzymes, protects cell surface antigens, and has synergistic enzyme effects.The lysis solution has complete lysis, short time, high cell viability, is suitable for large-scale production, is suitable for various cell types, retains cell stemness and secreted proteins, has simple components, low cost, adopts a three-enzyme synergistic mechanism + ion activation mechanism, does not need to add a chelating agent to inhibit ion enzyme activity, and solves the contradiction between rapid lysis and mild protection.The lysis solution is convenient to produce and is conducive to wide and rapid application in actual production and application.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology and relates to a high-efficiency lysis buffer for gelatin microcarriers based on a three-enzyme synergy and ion activation mechanism and its application. Background Technology

[0002] Three-dimensional cell culture technology can better simulate the microenvironment of cell growth in vivo, thus showing great application potential in tissue engineering, drug screening, and cell therapy. Currently, the mainstream approach for three-dimensional cell culture is to use non-degradable microcarriers (such as Cytodex series dextran microcarriers or plastic-based microcarriers), which provide high surface area support for cell attachment and proliferation. After three-dimensional culture, the key technological bottleneck for downstream applications lies in how to efficiently and gently harvest cells from the microcarriers while maintaining high cell viability and functional integrity.

[0003] For traditional non-degradable microcarriers, the mainstream method for harvesting cells from them is enzymatic digestion, primarily relying on trypsin (or its alternatives such as TrypLE) to degrade the adhesion proteins between cells and the carrier, causing cells to detach. Subsequent steps such as filtration or centrifugation are required to separate the cells from the microcarrier residue. However, this method has significant drawbacks: trypsin digestion is time-consuming, easily causing excessive damage to cell membranes and surface antigens, leading to decreased cell viability, low yield, and potentially affecting cell stemness, secretory function, and downstream therapeutic efficacy. Furthermore, the separation process is complex, easily resulting in cell loss and contamination risks, which is unfavorable for large-scale production.

[0004] In contrast, biodegradable microcarriers (such as gelatin or collagen-based microcarriers, for example, the 3DNovelGel series microcarriers; our company's products) allow for cell release through enzymatic degradation of the microcarrier itself, avoiding direct and vigorous enzymatic digestion of cells, thus achieving a gentler harvest. Existing technologies have patents reporting the use of collagenase to degrade gelatin microcarriers. For example, some existing technologies (such as CN121065065A) add metal chelating agents such as sodium citrate and calcium- and magnesium-free buffer solutions to the lysis buffer to aid cell dispersion or prevent aggregation. However, this approach ignores the fact that collagenase is essentially a calcium-dependent metalloproteinase; the addition of chelating agents inhibits enzyme activity, leading to prolonged lysis time. To compensate for the risks to cells from prolonged lysis, these methods have to add complex cell metabolism protectants such as arginine and vitamin C, which not only increases the cost of preparation but also fails to address the "rapid lysis" problem mechanistically. In contrast, this invention proposes for the first time a three-enzyme synergistic lysis system specifically designed for gelatin microcarriers that is trypsin-free, metal chelating agent-free, requires no complex chemical protectants, and is free of these agents.

[0005] This invention introduces Accutase as an alternative to trypsin. Accutase is a mild mixture of cell dissociation enzymes, containing proteolytic and collagenolytic enzymes, which can gently separate cell adhesion structures and intercellular connections while protecting the integrity of cell surface antigens, avoiding cell damage caused by traditional trypsin. The compound enzyme formulation of this invention is designed with a dual mechanism: on the one hand, collagenase (which mainly degrades the collagen backbone under calcium ion activation) and neutral protease synergistically and efficiently digest and completely degrade the gelatin microcarrier structure; on the other hand, Accutase enzyme specifically removes residual matrix connections on the cell surface, ensuring that cells gently detach from the degrading carrier. This compound formulation, through the synergistic effect of the three enzymes and calcium ion activation, achieves the dual effect of complete carrier lysis within 15 minutes and cell viability >98%, which is significantly superior to existing single or two-enzyme combinations. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to design and provide a technical solution for a high-efficiency lysis buffer for gelatin microcarriers based on a three-enzyme synergy and ion activation mechanism and its application.

[0007] The present invention is specifically implemented using the following technical solutions: The first aspect of this invention provides a high-efficiency lysis buffer for gelatin microcarriers, the high-efficiency lysis buffer for gelatin microcarriers comprising a complex enzyme and a buffer system, wherein the complex enzyme comprises at least one selected from collagenase, neutral protease, Accutase, Accumex, TrypLE, streptase, protease, dispersase, deoxyribonuclease I, lysozyme, papain, pepsin, chymotrypsin, cathepsin, clostridium protease, and plasmin.

[0008] Furthermore, the complex enzyme includes collagenase, neutral protease, and Accutase, with concentrations of 0.05%-0.2% (w / v) collagenase, 0.01%-0.1% (w / v) neutral protease, and 0.05%-0.5% (v / v) Accutase, respectively.

[0009] The collagenase of this invention rapidly disintegrates the carrier backbone under calcium ion activation, neutral protease synergistically cleaves cross-links, and Accutase enzyme specifically removes residual matrix connections on the cell surface. All three can achieve rapid lysis without the need for chemical protectants.

[0010] Furthermore, the buffer system includes HBSS, DPBS, PBS, EBSS, DMEM / F12 basal medium, RPMI1640 basal medium, MEM basal solution, Hepes buffered salt solution or other balanced salt solutions.

[0011] The preferred buffer is HBSS containing calcium and magnesium ions. This buffer system maintains the optimal activity of collagenase and enables rapid lysis within 10-20 minutes.

[0012] Furthermore, the pH of the buffer system is 7.0-7.4.

[0013] Furthermore, the buffer system contains calcium ions and magnesium ions, with a calcium ion concentration of 0.5-5 mM and a magnesium ion concentration of 0.1-2 mM.

[0014] A second aspect of the present invention provides a method for harvesting cells on gelatin microcarriers using a high-efficiency lysis buffer, comprising the following steps: (S.1) Allow the gelatin microcarriers containing cultured cells to settle naturally or centrifuge, remove the supernatant, and wash with buffer to obtain gelatin microcarriers containing cells. (S.2) Add the lysis buffer to the gelatin microcarrier containing cells and incubate for a period of time; (S.3) After the gelatin microcarriers have completely lysed, add the stop solution; (S.4) Centrifuge to remove the supernatant and obtain cells on the gelatin microcarrier.

[0015] Furthermore, in step S.2, the mass ratio of gelatin microcarrier to lysis buffer is 1 mg: 0.01-10 mL, and the incubation temperature is 4-42℃ for 5 min-48 h.

[0016] Furthermore, the termination solution in step S.3 is selected from at least one of whole culture medium, PBS containing 10% serum albumin or serum, and protease inhibitor, and the volume ratio of gelatin microcarrier to termination solution is 1 mg: 0.01-100 mL.

[0017] Furthermore, the cell types include 293T cells, HEK293 cells and their derivatives, MSCs, iPS cells, immune cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, primary cells or other adherent cells.

[0018] The third aspect of this invention provides an application of a high-efficiency gelatin microcarrier lysis buffer in cell therapy and regenerative medicine.

[0019] The present invention has the following beneficial effects: (1) The lysis buffer described in this invention is highly targeted and has high lysis efficiency. It is suitable for microcarriers made of collagen or gelatin, avoids direct exposure of cells to strong enzymes, protects cell surface antigens, and synergizes with enzyme effects.

[0020] (2) The lysis buffer described in this invention has a thorough lysis time, high cell viability, and is suitable for large-scale production. It is also suitable for multiple cell types and preserves cell stemness and secretory proteins.

[0021] (3) The present invention has simple components and low cost. It adopts the mechanism of three enzyme synergistic effect + ion activation, without adding chelating agents to inhibit enzyme activity and solves the contradiction between rapid lysis and mild protection.

[0022] (4) The pyrolysis solution described in this invention is easy to produce and is conducive to its rapid and wide-ranging application in actual production and use. Attached Figure Description

[0023] Figure 1 is a comparison of the lysis effects observed under a microscope using different enzyme combinations in Example 1 of the present invention; Figure 2 shows the observation of live cells under a fluorescence microscope in Embodiment 2 of the present invention. Detailed Implementation

[0024] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0025] Unless otherwise specified, the materials and reagents used in the following examples are commercially available. The gelatin microcarriers described below are 3D NovelGel microcarriers (products of our company).

[0026] The enzymes used in the embodiments of this invention are all commercially available products, wherein: Collagenase: Sigma-Aldrich Type I Collagenase (Catalog No. C0130, activity ≥125 CDU / mg solid).

[0027] Neutral protease: Sigma-Aldrich Roche Dispase II (catalog number 04942078001).

[0028] Accutase enzyme: Thermo Fisher (catalog number 00-4555-56).

[0029] If enzymes from other sources or batches are used, the concentration can be adjusted appropriately according to their activity units to achieve equivalent results.

[0030] Example 1: Comparison of lysis effects of different enzyme combinations 1. Reagent preparation: Solution A: 0.2% collagenase; Solution B: 0.2% neutral enzyme; Solution C: 0.1% collagenase + 0.05% neutral protease + 0.2% Accutase (dissolved in HBSS, pH 7.2); Solution D: (Comparative example, CN 121065065A): 0.1% collagenase + 10mM sodium citrate + 1mM L-arginine + 1mM vitamin C + 1mM sodium pyruvate (dissolved in calcium- and magnesium-free PBS, pH 7.2).

[0031] 2. Experimental methods: (1) Take 4 portions of gelatin microcarriers (products of our company) with the same batch number, 10mg each, and place them in centrifuge tubes.

[0032] (2) Add 1 mL of the above A, B, C and D lysis buffers respectively, vortex to mix, and incubate in a constant temperature shaker at 37℃. Take one sample at 10, 20, 30 and 40 minutes respectively, observe it with the naked eye first, then observe it under a microscope and take pictures to record the morphological changes and degree of lysis of the microcarrier.

[0033] (3) Results analysis: as shown in Table 1, Figure 1 As shown.

[0034] Table 1. Pyrolysis efficiency at different time points

[0035] Although a protective agent was added to group D, the cleavage efficiency was much lower than that of group C in this invention because sodium citrate chelated calcium ions, leading to a decrease in collagenase activity. Group C, through ion activation and synergistic effects of the three enzymes, achieved the fastest and most thorough cleavage without a protective agent.

[0036] Example 2: Harvesting 293T cells using an optimized lysis buffer formulation 1. Lysis buffer formulation: 0.1% collagenase, 0.05% neutral protease, 0.2% Accutase (v / v), dissolved in HBSS (pH 7.2).

[0037] 2. Transfer 20 mg of gelatin microcarriers adsorbed with 293T cells to a centrifuge tube, centrifuge at 1000×g for 2 min, aspirate the supernatant, and wash twice with HBSS.

[0038] 3. Add 2 mL of lysis buffer and incubate at 37°C for 25 min, gently mixing by pipetting every 5 min.

[0039] 4. After complete lysis, add 3 mL of complete culture medium to terminate the process.

[0040] 5. Centrifuge at 400×g for 5 minutes, discard the supernatant, and resuspend the cells.

[0041] 6. Methods for cell detection: (1) AO / PI double-staining fluorescence microscopy: 1) Take 100 μL of cell-containing microcarrier suspension and incubate with AO / PI staining solution for 30 min at a constant speed of 40 rpm.

[0042] 2) Centrifuge, remove supernatant, rinse twice with DPBS, add 2 mL lysis buffer, and observe the viable cells under a fluorescence microscope at 0, 15 and 30 minutes; the results are shown in Figure 2.

[0043] 3) As shown in Figure 2, when the stained cells were observed under a fluorescence microscope, the cell detachment rate from the microcarrier was >95%, the proportion of dead cells was <5%, and the cell viability rate was >95% as the lysis time increased. This indicates that the lysis buffer in this invention can harvest cells from the microcarrier in a gentle and efficient manner.

[0044] Example 3: The cleavage effect of the combination of collagenase and neutral protease 1. Lysis buffer formulation: 0.1% collagenase, 0.05% neutral protease (dissolved in HBSS, pH 7.2).

[0045] 2. Experimental methods: (1) Take 4 portions of gelatin microcarriers (products of our company) with the same batch number, each 10mg, and place them in 4 centrifuge tubes respectively.

[0046] (2) Add 1 mL of lysis buffer to each, vortex to mix, and incubate in a constant temperature shaker at 37℃. Take one sample at 0, 10, 20 and 30 minutes respectively, observe it with the naked eye first, then observe it under a microscope and take pictures to record the morphological changes and degree of lysis of the microcarrier.

[0047] (3) Results: At 20 minutes, the microcarrier partially disintegrated, with a lysis efficiency of approximately 70%; at 30 minutes, approximately 15% fragments remained, with a lysis efficiency of approximately 85%; the lysis time needed to be extended to 45 minutes to achieve >95% lysis. Compared to Group C (three enzymes) in Example 1, the lysis time was extended by approximately 50%, indicating poorer lysis completeness. This suggests that the lack of Accutase enzyme leads to insufficient cell dissociation and weakened synergistic effect.

[0048] Example 4: Lysis effect and cell harvesting of the combination of collagenase and Accutase 1. Lysis buffer formulation: 0.1% collagenase, 0.2% Accutase (dissolved in HBSS, pH 7.2).

[0049] 2. Experimental methods: (1) Take 20 mg of gelatin microcarriers that adsorb 293T cells, and add 2 mL of lysis buffer, and incubate at 37°C.

[0050] (2) Results: The lysis process was mild, and the cell viability rate was >92%; however, the carrier degradation was incomplete, with about 25% fragments remaining after 30 minutes, and the lysis efficiency was about 75%; it was necessary to extend the time to 50 minutes for basic dissolution. Compared with the three-enzyme combination in Example 1, the lysis time was extended by about 2 times, and the yield was only about 85%. This indicates that the lack of neutral protease leads to insufficient cleavage of non-collagenous links, affecting the complete degradation of the carrier.

[0051] Example 5: The effect of changing the buffer system on the pyrolysis effect 1. Lysis buffer formulation: 0.1% collagenase, 0.05% neutral protease, 0.2% Accutase, dissolved in PBS (without calcium and magnesium ion supplementation, pH 7.2) control HBSS group.

[0052] 2. Take 10 mg of gelatin microcarrier, add 1 mL of lysis buffer, vortex to mix, and incubate in a constant temperature shaker at 37℃. Take one sample at each of the following times: 0, 10, 20, and 30 minutes. First observe with the naked eye, then observe and photograph under a microscope to record the morphological changes and degree of lysis of the microcarrier.

[0053] 3. Results: The lysis efficiency of the PBS group was approximately 80% after 20 minutes and approximately 92% after 30 minutes, requiring 40 minutes to achieve >98% complete lysis; the HBSS group (such as group C in Example 1) achieved >98% lysis after 30 minutes. The PBS group, due to the lack of calcium ions and the potential competition from trace amounts of phosphate ions, showed a significant decrease in collagenase activity and a prolongation of lysis time by approximately 30%. This indicates that HBSS (containing calcium and magnesium) is a preferred buffer system (calcium ion concentration of 0.5-5 mM and magnesium ion concentration of 0.1-2 mM), which can enhance collagenase activity and achieve faster lysis.

[0054] Example 6: Harvesting mesenchymal stem cells using optimized lysis buffer 1. Lysis buffer formulation: 0.1% collagenase, 0.05% neutral protease, 0.2% Accutase, dissolved in HBSS (pH 7.2).

[0055] 2. Transfer 30 mg of gelatin microcarriers adsorbed with human bone marrow-derived mesenchymal stem cells to a centrifuge tube, centrifuge at 1000×g for 3 min, aspirate the supernatant, and wash twice with HBSS.

[0056] 3. Add 3 mL of lysis buffer and incubate at 37°C for 30 min, gently mixing by pipetting every 5 min.

[0057] 4. After complete lysis, add 5 mL of full culture medium containing 10% serum to terminate the process.

[0058] 5. Centrifuge at 400×g for 5 minutes, discard the supernatant, and resuspend the cells.

[0059] 6. Cell detection: AO / PI double staining fluorescence microscopy showed that the cell detachment rate was >96% and the viability rate was >94%, indicating that the lysis buffer of this invention can efficiently and gently harvest mesenchymal stem cells from gelatin microcarriers.

[0060] Example 7: Harvesting human embryonic stem cells (hESCs) using optimized lysis buffer 1. Lysis buffer formulation: 0.08% collagenase, 0.03% neutral protease, 0.3% Accutase, dissolved in HBSS (pH 7.2).

[0061] 2. Transfer 15 mg of gelatin microcarriers adsorbed with human embryonic stem cells (hESCs) to a centrifuge tube, centrifuge at 800×g for 2 min, aspirate the supernatant, and wash twice with HBSS.

[0062] 3. Add 1.5 mL of lysis buffer and incubate at 37 °C for 20 min, gently mixing by pipetting every 5 min.

[0063] 4. After complete lysis, add 2 mL of hESC full medium containing ROCK inhibitor to terminate the process.

[0064] 5. Centrifuge at 300×g for 4 min, discard the supernatant, and resuspend the cells.

[0065] 6. Cell detection: AO / PI staining and immunofluorescence of multifunctional markers such as Oct4 and Nanog. Results showed a cell detachment rate >95%, viability >96%, and no significant decrease in the expression of multifunctional markers. This indicates that the lysis buffer of this invention is particularly suitable for preserving the multifunctionality of human embryonic stem cells (hESCs).

Claims

1. A high-efficiency lysis buffer for gelatin microcarriers, characterized in that, The high-efficiency lysis buffer for gelatin microcarriers includes a complex enzyme and a buffer system. The complex enzyme includes at least one of collagenase, neutral protease, Accutase, Accumex, TrypLE, streptase, protease, dispersase, deoxyribonuclease I, lysozyme, papain, pepsin, chymotrypsin, cathepsin, clostridium protease, and plasmin.

2. The high-efficiency lysis buffer for gelatin microcarriers as described in claim 1, characterized in that, The complex enzyme includes collagenase, neutral protease, and Accutase, with concentrations of 0.05%-0.2% (w / v) collagenase, 0.01%-0.1% (w / v) neutral protease, and 0.05%-0.5% (v / v) Accutase, respectively.

3. The high-efficiency lysis buffer for gelatin microcarriers as described in claim 1, characterized in that, The buffer system includes HBSS, DPBS, PBS, EBSS, DMEM / F12 basal medium, RPMI1640 basal medium, MEM basal solution, Hepes buffered salt solution or other balanced salt solutions.

4. The high-efficiency lysis buffer for gelatin microcarriers as described in claim 3, characterized in that, The pH of the buffer system is 7.0-7.

4.

5. The high-efficiency lysis buffer for gelatin microcarriers as described in claim 3, characterized in that, The buffer system contains calcium ions and magnesium ions, with a calcium ion concentration of 0.5-5 mM and a magnesium ion concentration of 0.1-2 mM.

6. A method for harvesting cells on gelatin microcarriers using a high-efficiency lysis buffer for gelatin microcarriers as described in any one of claims 1-5, characterized in that, Includes the following steps: (S.1) Allow the gelatin microcarriers containing cultured cells to settle naturally or centrifuge, remove the supernatant, and wash with buffer to obtain gelatin microcarriers containing cells. (S.2) Add the lysis buffer to the gelatin microcarrier containing cells and incubate for a period of time; (S.3) After the gelatin microcarriers have completely lysed, add the stop solution; (S.4) Centrifuge to remove the supernatant and obtain cells on the gelatin microcarrier.

7. The method for harvesting cells on gelatin microcarriers as described in claim 6, characterized in that, In step S.2, the mass ratio of gelatin microcarrier to lysis buffer is 1 mg: 0.01-10 mL, and the incubation temperature is 4-42℃ for 5 min-48 h.

8. The method for harvesting cells on gelatin microcarriers as described in claim 6, characterized in that, The termination solution in step S.3 is selected from at least one of the following: whole culture medium, PBS containing 10% serum albumin or serum, and protease inhibitor. The volume ratio of gelatin microcarrier to termination solution is 1 mg: 0.01-100 mL.

9. The method for harvesting cells on gelatin microcarriers as described in claim 6, characterized in that, The cell types include 293T cells, HEK293 cells and their derivatives, MSCs, iPS cells, immune cells, fibroblasts, epithelial cells, endothelial cells, hepatocytes, primary cells or other adherent cells.

10. The application of the gelatin microcarrier high-efficiency lysis buffer as described in any one of claims 1-5 in cell therapy and regenerative medicine.

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